Desaturases are a class of enzymes critical in the production of long-chain polyunsaturated fatty acids. Polyunsaturated fatty acids (PUFAs) play many roles in the proper functioning of all life forms. For example, PUFAs are important components of the plasma membrane of a cell, where they are found in the form of phospholipids. PUFAs also are precursors to mammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins. Additionally, PUFAs are necessary for the proper development of the infant brain, as well as for tissue formation and repair in mature mammals. In view of the biological significance of PUFAs, attempts are being made to produce them in an efficient manner.
A number of enzymes, most notably desaturases and elongases, are involved in PUFA biosynthesis (see FIG. 1). Elongases catalyze the addition of a 2-carbon unit to a fatty acid substrate. Thus, for example, an elongase (generically designated “elo” in FIG. 1) catalyzes the conversion of γ-linolenic acid (18:3n-6) to dihomo-γ-linolenic acid (20:3n-6), as well as the conversion of stearidonic acid (18:4n-3) to eicosatetraenoic acid (20:4n-3), etc.
Desaturases catalyze the introduction of unsaturations (i.e., double bonds) between carbon atoms within the fatty acid alkyl chain of the substrate. Thus, for example, linoleic acid (18:2n-6) is produced from oleic acid (18:1n-9) by the action of a Δ12-desaturase. Similarly, γ-linolenic acid (18:3n-6) is produced from linoleic acid by the action of a Δ6-desaturase.
Throughout the present application, PUFAs will be unambiguously identified using the “omega” system of nomenclature favored by physiologists and biochemists, as opposed to the “delta” system or I.U.P.A.C. system normally favored by chemists. In the “omega” system, a PUFA is identified by a numeric designation of the number of carbons in the chain. This is followed by a colon and then another numeric designation of the number of unsaturations in the molecule. This is then followed by the designation “n−x,” where x is the number of carbons from the methyl end of the molecule where the first unsaturation is located. Each subsequence unsaturation (where there is more than one double bond) is located 3 addition carbon atoms toward the carboxyl end of the molecule. Thus, the PUFAs described herein can be described as being “methylene-interrupted” PUFAs. Where some other designation is required, deviations from the “omega” system will be noted.
Where appropriate, the action of the desaturase enzymes described herein will also be identified using the “omega” system. Thus, an “omega-3” desaturase catalyzes the introduction of a double bond between the two carbons at positions 3 and 4 from the methyl end of the substrate. However, in many instances, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the substrate. Thus, as shown in FIG. 1, a Δ9-desaturase catalyzes the introduction of a double bond between the two carbons at positions 9 and 10 from the carboxyl end of the substrate. In short, where the term “omega” is used, the position on the substrate is being designated relative to the methyl terminus; where the term “delta” is used, the position on the substrate is being designated relative to the carboxyl terminus.
It must be noted that mammals cannot desaturate fatty acid substrates beyond the Δ9 position (i.e., beyond 9 carbon atoms distant from the carboxyl terminus). Thus, for example, mammals cannot convert oleic acid (18:1n-9) into linoleic acid (18:2n-6); linoleic acid contains an unsaturation at position Δ12. Likewise, α-linolenic acid (18:3n-3) (having unsaturations at Δ12 and Δ15) cannot be synthesized by mammals. However, for example, mammals can convert α-linolenic acid into stearidonic acid (18:4n-3) by the action of a Δ6-desaturase. (See FIG. 1. See also PCT publication WO 96/13591; The FASEB Journal, Abstracts, Part I, Abstract 3093, page Δ532 (Experimental Biology 98, San Francisco, Calif., Apr. 18-22, 1998); and U.S. Pat. No. 5,552,306.)
Still referring to FIG. 1, in mammals, fungi, and algae, the stearidonic acid so formed is converted into eicosatetraenoic acid (20:4n-3) by the action of an elongase. This PUFA can then be converted to eicosapentaenoic acid (20:5n-3) by a Δ5-desaturase. Eicosapentaenoic acid can then, in turn, be converted to ω3-docosapentaenoic acid (22:5n-3) by an elongase.
Other eukaryotes, including fungi and plants, have enzymes that desaturate fatty acid substrates at carbon Δ12 (see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and at carbon delta-15 (see PCT publication WO 93/11245). The major polyunsaturated fatty acids of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid or α-linolenic acid. In view of these difficulties, there remains a significant need to isolate genes involved in PUFA synthesis. Ideally, these genes would originate from species that naturally produce fatty acids that are not produced naturally in mammals. These genes could then be expressed in a microbial, plant, or animal system, which would thereby be altered to produce commercial quantities of one or more PUFAs. Thus, there is a definite need for novel Δ12- and Δ17-desaturase enzymes, the respective genes encoding these enzymes, as well as recombinant methods of producing these enzymes. Additionally, a need exists for oils containing levels of PUFAs beyond those naturally present. Access to such Δ12- and Δ17-desaturase enzymes allows for the production of large amounts of PUFAs that cannot be synthesized de novo in mammals. These PUFAs can be used as pharmaceutical agents and/or nutritional supplements.
All patents, patent publications and priority documents cited herein are hereby incorporated by reference in their entirety.